Phthalate esters have been used extensively as plasticizers of synthetic polymers. Recent studies have revealed that these esters induce atrophy of the testis, although its pathogenesis remains unknown. The present study describes the possible involvement of oxidative stress in the pathogenesis of atrophy of the rat testis induced by di(2-ethylhexyl)phthalate (DEHP). Biochemical and immunohistochemical analysis revealed that oral administration of DEHP increased the generation of reactive oxygen species, with concomitant decrease in the concentration of glutathione and ascorbic acid in the testis, and selectively induced apoptosis of spermatocytes, thereby causing atrophy of this organ. Oxidative stress was selectively induced in germ cells, but not in Sertoli cells, treated with mono(2-ethylhexyl)phthalate (MEHP), a hydrolysed metabolite of DEHP. Furthermore, MEHP selectively induced the release of cytochrome c from mitochondria of the testis. These results indicate that oxidative stress elicited by MEHP principally injured mitochondrial function and induced the release of cytochrome c, thereby inducing apoptosis of spermatocytes and causing atrophy of the testis.